There are different methods for identification of materials including macroscopic, chemical and microscopic identification among others. Microscopic identification is a technique that uses a microscope to identify characteristic features of living organisms, parts of an organism, cells or sub-cellular organs, as well as minerals or other non-living materials. The importance of microscopy resides in the ability to clearly identify differences between organisms or their parts by focusing on specific characteristics or diagnostic structures. Microscopy relies on dependable, readily available reagents as clearing agent and mounting solutions, optionally used in conjunction with stains in order to visualize the materials under the slide.
The general microscopy procedure for specimens derived from living organisms comprises mounting a small sample of the tissue to be analyzed in a solvent solution and observing it under the microscope. In many cases the cell contents obscure the tissues, making it difficult to identify characteristic features. Differences in refractive index within the specimen prohibit visualization of deeper visual planes, and occlude detail from observation. In these cases a clearing solution is applied in order to improve the transparency of the specimen, allowing one to visualize multiple vertical layers of the specimen without careful sectioning or remounting. This increased transparency and improved clarity allows the microscope user to visualize across a full range of vertical planes in the sample, allowing the user to select interesting focal planes by adjusting the focus.
A valuable and almost universally used clearing agent for microscopy is acidified chloral hydrate glycerol solution (chloral hydrate solution acidified with hydrochloric acid), also known as Hertwig's solution. Acidified chloral hydrate solution is used in botanical microscopy, mycology, entomology, histology, mineralogy, food science, quality control, forensics, nematology, archeology, paleontology, virology, immunology, microscopy including but not limited to differential interference contrast microscopy, electron microscopy, fluorescence microscopy, confocal microscopy, and other related applications of microscopy and optics. Chloral hydrate when applied to botanical samples dissolves cellular contents and intercellular substances thus allowing cell walls and shapes of the cells to be easily observed. Chloral hydrate solution has a high refractive index, which improves its transparency over media with lower refractive indices (such as water).
Many Pharmacopeias contain published protocols for microscopic authentication analyses of herbal preparations using acidified chloral hydrate as the clearing agent. Consequently, chloral hydrate has become the industry standard and an important reagent required on a daily basis for many laboratories focused on quality assessment of herbal products. For example, in botanical samples, chloral hydrate is used to assist in the identification of cells with suberized cell walls, fibers (an elongated cell with thick cell walls at maturity that gives strength and support to the plant tissue), vessel elements (a tube-like series of cells with wide lumen and perforated walls), trichomes (hair-like structures that project from the epidermal surface of the leaves, flowers and stems), stoma (pores in the epidermis of the leaf through which the plant exchanges gases) and pollen.
Chloral hydrate, the key component in acidified chloral hydrate solution, is considered under US law to be a narcotic hypnotic, and as such is a DEA (Drug Enforcement Administration) scheduled substance, requiring DEA approval and compliance in order to purchase and/or possess it. This has precluded scientists from being able to purchase this reagent; and in particular has precluded academic institutions (elementary schools, middle schools, high schools, colleges and universities) from purchasing this almost universal reagent that is required for preparing slides across all disciplines—from plant science, to biological sciences, to medical sciences and more. Furthermore, maintaining DEA compliance is a costly, tedious, and time-consuming process.
Thus, a major disadvantage of using acidic chloral hydrate-glycerol solution is that chloral hydrate is a controlled substance and requires a special permit in order to purchase, possess, or use in the US. Therefore cost-effective, readily available and unregulated replacements for acidified chloral hydrate solution are needed as clearing and mounting agents for microscopy.